Osteoimplant and method for its manufacture

ABSTRACT

The invention relates to an osteoimplant fabricated from a solid aggregate of bone derived elements possessing chemical linkages between their adjacent surface-exposed collagen. Also described are various other components which can be incorporated into the bone implant material such as bone-growth inducing substances; and a method of manufacture.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention relates to an osteoimplant for use in the repair,replacement and/or augmentation of various portions of animal or humanskeletal systems and to a method for manufacturing the osteoimplant.More particularly, this invention relates to an osteoimplant made up ofa solid aggregate of bone-derived elements that are bonded to each otherthrough chemical linkages formed between their surface-exposed collagen.

2. Description of the Related Art

The use of autograft bone, allograft bone or xenograft bone is wellknown in both human and veterinary medicine. See Stevenson et al.,Clinical Orthopedics and Related Research, 323, pp. 66-74 (1996). Inparticular, transplanted bone is known to provide support, promotehealing, fill bony cavities, separate bony elements such as vertebralbodies, promote fusion and stabilize the sites of fractures. Morerecently, processed bone has been developed into shapes for use in newsurgical applications, or as new materials for implants that werehistorically made of non-biologically derived materials.

U.S. Pat. No. 4,678,470 describes a non-layered bone grafting materialproduced from bone by a process which includes tanning withglutaraldehyde. The bone may be pulverized, used as a large block ormachined into a precise shape. The tanning stabilizes the material andalso renders it non-antigenic. The bone material may also bedemineralized.

Collagen is a naturally occurring structural biomaterial and is acomponent of connective tissues, including bone, in all vertebratespecies. Native collagen is a glycine-rich chain of amino acids arrangedin a triple helix and can be crosslinked by a variety of procedures.

Tissue transglutaminase is described as being effective at increasingadhesive strength at a cartilage-cartilage interface. See Jurgensen, K.,et al., The Journal of Bone and Joint Surgery, 79-A (2), 185-193 (1997).

U.S. Pat. No. 5,507,813 describes a surgically implantable sheet formedfrom elongate bone particles, optionally demineralized, containingbiocompatible ingredients, adhesives, fillers, plasticizers etc.

U.S. Pat. No. 4,932,973 discloses an artificial organic bone matrix withholes or perforations extending into the organic bone matrix. The holesor perforations are indicated to be centers of cartilage and boneinduction following implantation of the bone matrix.

U.S. Pat. No. 4,394,370 discloses a one-piece sponge-like bone graftmaterial fabricated from fully demineralized bone powder or microparticulate bone, and reconstituted collagen. The sponge-like graft isoptionally crosslinked with glutaraldehyde.

Another one-piece porous implant is described in U.S. Pat. No.5,683,459. The implant is made up of a biodegradable polymericmacrostructure, which is structured as an interconnecting open cellmeshwork, and a biodegradable polymeric microstructure composed ofchemotactic ground substances such as hyaluronic acid.

SUMMARY OF THE INVENTION

The present invention provides an osteoimplant which, due to chemicallinkages formed between the surface-exposed collagen of adjacentpartially demineralized bone elements from which the osteoimplant ismanufactured, exhibits good mechanical strength, is biocompatible and,in a preferred embodiment, through its bone healing activity and abilityto contain bone-growth inducing substances, can promote and/oraccelerate new bone growth.

It is therefore an object of the present invention to provide anosteoimplant made up of a solid aggregate of bone-derived elements,adjacent bone-derived elements being bonded to each other throughchemical linkages between their surface-exposed collagen, and whichpossesses good mechanical strength and biocompatibility.

It is another object of this invention to provide an osteoimplant whichcan optionally include another component such as a reinforcing particleor fiber, fillers, bone-growth inducing substances such asmedically/surgically useful substances, and combinations thereof.

It is another object of the invention to provide an osteoimplantpossessing a network of pores, perforations, apertures, channels orspaces which permits and encourages penetration by endogenous andexogenous bone healing materials and blood supply, and simultaneouslyprovides a means for incorporating one or more bone healing substances.

It is yet a further object of the present invention to provide anosteoimplant which can be fashioned into a variety of shapes and sizeswhich are not limited by constraints imposed by the size and/or types ofdonor bone which are available for construction of the osteoimplant.

It is also an object of the invention to provide a method ofmanufacturing which will provide a strong, biocompatible osteoimplant ofany size and/or shape for implantation.

In keeping with these and other objects of the invention, there isprovided an osteoimplant which comprises a solid aggregate ofbone-derived elements with adjacent bone-derived elements being bondedto each other through chemical linkages between their surface-exposedcollagen.

Further in keeping with the invention, there is provided a method forthe manufacture of an osteoimplant which comprises providing a quantityof bone-derived elements presenting surface-exposed collagen and formingchemical linkages between the surface-exposed collagen to bond theelements into a solid aggregate.

The osteoimplant of the present invention possesses a significantadvantage over the prior art in its ability to be biocompatible,non-antigenic and to provide good mechanical strength.

Another important advantage of the osteoimplant herein over prior artimplants lies in its ability to function as a carrier for, andeffectively diffuse, one or more bone-growth inducing substances thatpromote new bone growth and/or accelerate healing.

The term "osteogenic" as used herein shall be understood to refer to theability of a substance to induce new bone formation via theparticipation of living cells from within the substance.

The term "osteoconductive" as used herein shall be understood to referto the ability of a substance or material to provide biologically inertsurfaces which are receptive to the growth of new host bone.

The term "osteoinductive" as used herein shall be understood to refer tothe ability of a substance to recruit cells from the host which have thepotential for repairing bone tissue.

Use of the expression "bone-derived elements" shall be understood torefer to pieces of bone in any variety of sizes, thicknesses andconfigurations including particles, fibers, strips, thin to thicksheets, etc., which can be obtained by milling, slicing, cutting ormachining whole bone.

The expression "surface-exposed collagen" shall be understood to referto the result obtained by demineralizing the aforementioned bone-derivedelements, the demineralization ranging from substantially complete (inwhich case the bone-derived elements are primarily collagen) to partialor superficial (in which case only the surfaces of the bone-derivedelements present exposed collagen). Partial or superficialdemineralization produces bone-derived elements having a surface bindingregion, namely, exposed collagen while retaining a strengthening region,namely, the unaffected mineralized region of the bone-derived elements.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments are described below with reference to the drawingswherein:

FIG. 1 is a cross-sectional view of bone from the diaphyseal regionwhich has been sliced longitudinally into several cortical bone sheets;

FIG. 2 is an enlarged perspective view of an osteoimplant of theinvention possessing sheets of partially demineralized bone at theirsurface and an interior made up of mineralized or partiallydemineralized bone;

FIG. 3 is a view of a human femur showing an osteoimplant of theinvention, as shown in FIG. 3A, fashioned as a femoral bone replacement;

FIG. 4 is a partial view of the human vertebral column showing adisc-shaped osteoimplant of the invention installed at an intervertebralsite;

FIGS. 5 and 5A are views of a human skull showing an osteoimplant of theinvention fashioned as a parietal bone replacement;

FIG. 6 is an enlarged perspective view of an osteoimplant of theinvention possessing alternating layers of bone sheets and cubes withchannels between the cubes.

FIG. 7 is a partial view of the human vertebral column showinginstallation of the osteoimplant of FIG. 6 at a posterolateralintertransverse process fusion site;

FIG. 8 is a perspective view depicting separated layers of anosteoimplant of the invention; and,

FIG. 8A depicts an osteoimplant of the invention possessing layers ofbone sheets bonded together via chemical bonds formed by catalysis withtissue transglutaminase, as shown in FIG. 8.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The osteoimplant of the present invention comprises a solid aggregate ofbone-derived elements having chemical linkages between theirsurface-exposed collagen molecules thus bonding adjacent bone elementsto each other. In order to expose the collagen located on the outersurface of bone, the bone elements must be at least partiallydemineralized. Demineralization methods remove the mineral component ofbone employing acid solutions. Such methods as used by the presentinvention are well known in the art, see for example, Reddi et al.,Proc. Nat. Acad. Sci. 69, pp1601-1605 (1972), incorporated herein byreference. The strength of the acid solution, the shape of the bone andthe duration of the demineralization treatment will determine the extentof demineralization. Reference in this regard may be made toLewandrowski et al., J. Biomed Materials Res, 31, pp365-372 (1996), alsoincorporated herein by reference. The sources for the bone-derivedelements herein include cortical and cancellous bone and are preferablyallogenic but also include xenogenic sources such as bovine and porcinebone.

When prepared from bone-derived elements that are only superficiallydemineralized, the osteoimplant herein will tend to possess a fairlyhigh compression strength, e.g., one approaching that of natural bone.Accordingly, when an osteoimplant exhibiting relatively high compressionstrength is desired, e.g., on the order of from about 10 to about 200MPa, and preferably from about 20 to about 100 MPa, it is necessary toemploy bone-derived elements which retain a high proportion of theiroriginal mineral content or, stated another way, which have only beensuperficially demineralized.

In addition to containing bone-derived elements, the osteoimplant ofthis invention can optionally possess one or more other components suchas reinforcing particles, fibers, fillers, bone-growth inducingsubstances, adhesives, plasticizers, flexibilizing agents, hydrationfacilitating agents, biostatic/biocidal agents, substances impartingradiopacity, metallic meshes and the like. Examples of reinforcingparticles include fully mineralized cortical and cancellous bone, andpartially demineralized cortical and cancellous bone in any form,including particles, sheets and shaped bone pieces; graphite orpyrolytic carbon. Examples of fillers include mineral material such ashydroxyapatite, tricalcium phosphate and other calcium salts, bonepowder, fully mineralized and partially or fully demineralized corticaland cancellous bone in any form, including particles such asdemineralized bone powder (or "demineralized bone matrix" as it may alsobe called) sheets and shaped bone pieces, graphite or pyrolytic carbon,bioglass or other bioceramic or natural or synthetic polymers, e.g.,bioabsorbable polymers such as polyglycolide, polylactide,glycolide-lactide copolymer, and the like, and nonbioabsorbablematerials such as starches, polymethyl methacrylate,polytetrafluoroethylene, polyurethane, polyethylene and nylon. Suitableplasticizers, flexibilizing agents and hydration facilitating agents,include liquid polyhydroxy compounds such as glycerol, monacetin,diacetin, and mixtures thereof. Suitable biostatic/biocidal agentsinclude antibiotics, povidone, sugars, and mixtures thereof; suitablesurface agents include the biocompatible nonionic, cationic, anionic andamphoteric surfactants, and mixtures thereof. The osteoimplant can alsopossess bone-growth inducing substances which include any of a varietyof medically and/or surgically useful substances which are describedbelow.

The osteoimplant can possess one or more cavities which, if desired, cancommunicate with the surface of the implant through pores, apertures,perforations or channels provided for this purpose and ranging inaverage diameter from a few microns to several millimeters. Suchcavities and their associated pores, apertures, perforations, andchannels can be partially or completely filled with one or moremedically/surgically useful substances which promote or accelerate newbone growth or bone healing due, e.g., to some osteogenic,osteoconductive and/or osteoconductive effect. Useful substances of thiskind which can be incorporated into the osteoimplant of this inventioninclude, e.g., collagen, insoluble collagen derivatives, etc., andsoluble solids and/or liquids dissolved therein, e.g., antiviral agents,particularly those effective against HIV and hepatitis; antimicrobialsand/or antibiotics such as erythromycin, bacitracin, neomycin,penicillin, polymyxin B, tetracyclines, viomycin, chloromycetin andstreptomycins, cefazolin, ampicillin, azactam, tobramycin, clindamycinand gentamicin, etc.; biocidal/biostatic sugars such as dextrose,glucose, etc.; amino acids, peptides, vitamins, inorganic elements,co-factors for protein synthesis; hormones; endocrine tissue or tissuefragments; synthesizers; enzymes such as collagenase, peptidases,oxidases, etc.; polymer cell scaffolds with parenchymal cells;angiogenic drugs and polymeric carriers containing such drugs; collagenlattices; antigenic agents; cytoskeletal agents; cartilage fragments,living cells such as chondrocytes, bone marrow cells, mesenchymal stemcells, natural extracts, tissue transplants, bone, demineralized bone,autogenous tissues such as blood, serum, soft tissue, bone marrow, etc.;bioadhesives, bone morphogenic proteins (BMPs), transforming growthfactor (TGF-beta), insulin-like growth factor (IGF-1); growth hormonessuch as somatotropin; bone digesters; antitumor agents;immunosuppressants; angiogenic agents such as basic fibroblast growthfactor (bFGF); permeation enhancers, e.g., fatty acid esters such aslaureate, myristate and stearate monoesters of polyethylene glycol,enamine derivatives, alpha-keto aldehydes, etc.; and, nucleic acids.These and similar medically/surgically useful substances can beincorporated into the osteoimplant of this invention or any of itsconstituent bone-derived elements or other components during any stageof the assembly of the implant. Suitable methods of incorporationinclude coating, immersion saturation, packing, etc. The amounts ofmedically/surgically useful substances utilized can vary widely withoptimum levels being readily determined in a specific case by routineexperimentation.

Osteoimplants of any desirable size and/or configuration can beprovided, e.g., by machining or other mechanical shaping operations suchas press-molding. Computerized modeling of a specific implant followedby computerized control of the shaping of the implant can be used toprovide an intricately shaped osteoimplant which is custom-fitted to theintended site of application with great precision.

Where the invention comprises aggregates of elongate bone-derivedelements which, in appearance can be described as filaments, fibers,threads, slender or narrow strips, etc., an osteoimplant can be formedfrom these elements by a variety of methods. For example, forming asolution or slurry in a suitable medium which can comprise thecrosslinking agent, and any proportion of the elongate bone-derivedelements being partially or fully demineralized, and fully mineralized.This solution can be formed into an osteoimplant of any shape accordingto the configuration of a mold into which it is poured. The mold ispreferably shaped as a bone or section thereof, or as an implant forgrafting. Once contained in a mold, the solution of bone-derivedelements can be solidified into a solid osteoimplant by knowntechniques.

It is within the scope of the invention to supplement or increase theshape-retaining and/or mechanical strength characteristics of theosteoimplant, e.g., by the addition of mechanical fasteners such aspins, screws, dowels, etc., which can be fabricated from natural orsynthetic materials and bioabsorbable as well as nonbioabsorbalematerials, by the use of laser tissue welding or ultrasonic bonding, andso forth. In those embodiments of the osteoimplant which are assembledfrom relatively large bone-derived elements such as sheets, suchelements can be provided with mechanically interengaging features, e.g.,tongue-and-groove or mortise-and-tenon features, which facilitate theirassembly into the final product and/or to fix the elements to each otherin a more secured fashion.

The osteoimplant herein is intended to be applied at a bone defect site,e.g., one resulting from injury, defect brought about during the courseof surgery, infection, malignancy or developmental malformation. Theosteoimplant, suitably sized and shaped as required, can be utilized asa graft or replacement in a wide variety of orthopaedic, neurosurgicaland oral and maxillofacial surgical procedures such as the repair ofsimple and compound fractures and non-unions, external and internalfixations, joint reconstructions such as arthrodesis, generalarthroplasty, cup arthroplasty of the hip, femoral and humeral headreplacement, femoral head surface replacement and total jointreplacement, repairs of the vertebral column including spinal fusion andinternal fixation, tumor surgery, e.g., deficit filling, discectomy,laminectomy, excision of spinal cord tumors, anterior cervical andthoracic operations, repair of spinal injuries, scoliosis, lordosis andkyphosis treatments, intermaxillary fixation of fractures, mentoplasty,temporomandibular joint replacement, alveolar ridge augmentation andreconstruction, onlay bone grafts, implant placement and revision, sinuslifts, etc. Specific bones which can be repaired or replaced with theosteoimplant herein include the ethmoid, frontal, nasal, occipital,parietal, temporal, mandible, maxilla, zygomatic, cervical vertebra,thoracic vertebra, lumbar vertebra, sacrum, rib, sternum, clavicle,scapula, humerus, radius, ulna, carpal bones, metacarpal bones,phalanges, ilium, ischium, pubis, femur, tibia, fibula, patella,calcaneus, tarsal, and metatarsal bones.

The method of manufacturing the osteoimplant of the present inventioncomprises providing a quantity of bone-derived elements initiallypresenting surface-exposed collagen and subsequently forming chemicallinkages between the surface-exposed collagen of adjacent bone-derivedelements to bond the elements into a solid aggregate. These chemicallinkages can be formed employing a variety of known methods includingchemical reaction, the application of energy such as radiant energy,which includes irradiation by UV light or microwave energy, dryingand/or heating and dye-mediated photo-oxidation; dehydrothermaltreatment in which water is slowly removed while the bone tissue issubjected to a vacuum; and, enzymatic treatment to form chemicallinkages at any collagen-collagen interface. The preferred method offorming chemical linkages is by chemical reaction.

Chemical crosslinking agents include those that contain bifunctional ormultifunctional reactive groups, and which react with functional groupson amino acids such as epsilon-amine functional group of lysine orhydroxy-lysine, or the carboxyl functional groups of aspartic andglutamic acids. By reacting with multiple functional groups on the sameor different collagen molecules, the reacting chemical crosslinkingagent forms a reinforcing cross-bridge.

Suitable chemical crosslinking agents include: mono- and dialdehydes,including glutaraldehyde and formaldehyde; polyepoxy compounds such asglycerol polyglycidyl ethers, polyethylene glycol diglycidyl ethers andother polyepoxy and diepoxy glycidyl ethers; tanning agents includingpolyvalent metallic oxides such as titanium dioxide, chromium dioxide,aluminum dioxide, zirconium salt, as well as organic tannins and otherphenolic oxides derived from plants; chemicals for esterification ofcarboxyl groups followed by reaction with hydrazide to form activatedacyl azide functionalities in the collagen; dicyclohexyl carbodiimideand its derivatives as well as other heterobifunctional crosslinkingagents; hexamethylene diisocyanate; sugars, including glucose, will alsocrosslink collagen.

Glutaraldehyde crosslinked biomaterials have a tendency to over-calcifyin the body. In this situation, should it be deemed necessary,calcification-controlling agents can be used with aldehyde crosslinkingagents. These calcification-controlling agents include: dimethylsulfoxide (DMSO), surfactants, diphosphonates, aminooleic acid, andmetallic ions, for example ions of iron and aluminum. The concentrationsof these calcification-controlling agents can be determined by routineexperimentation by those skilled in the art.

Chemical crosslinking involves exposing the bone-derived elementspresenting surface-exposed collagen to the chemical crosslinking agent,either by placing the elements in a solution of the chemicalcrosslinking agent, or by exposing them to the vapors of the chemicalcrosslinking agent under conditions appropriate for the particular typeof crosslinking reaction. Such conditions include: an appropriate pH andtemperature, and for times ranging from minutes to days, depending uponthe level of crosslinking desired, and the activity of the chemicalcrosslinking agent. The osteoimplant is then washed to remove allleachable traces of the chemical.

When enzymatic treatment is employed, useful enzymes include those knownin the art which are capable of catalyzing crosslinking reactions onproteins or peptides, preferably collagen molecules, e.g.,transglutaminase as described in Jurgensen et al., The Journal of Boneand Joint Surgery, 79-A (2), 185-193 (1997), herein incorporated byreference.

Formation of chemical linkages can also be accomplished by theapplication of energy. One way to form chemical linkages by applicationof energy is to use methods known to form highly reactive oxygen ionsgenerated from atmospheric gas, which in turn, promote oxygen crosslinksbetween surface-exposed collagen. Such methods include using energy inthe form of ultraviolet light, microwave energy and the like. Anothermethod utilizing the application of energy is a process known asdye-mediated photo-oxidation in which a chemical dye under the action ofvisible light is used to crosslink surface-exposed collagen.

Another method for the formation of chemical linkages is bydehydrothermal treatment which uses combined heat and the slow removalof water, preferably under vacuum, to achieve crosslinking of thebone-derived elements. The process involves chemically combining ahydroxy group from a functional group of one collagen molecule and ahydrogen ion from a functional group of another collagen moleculereacting to form water which is then removed resulting in the formationof a bond between the collagen molecules.

Referring to the drawings, as shown in FIG. 1, the cortical portion ofbone 10 taken from the diaphyseal region is cut into cortical bonesheets 11 of varying width by slicing the bone longitudinally. Ifdesired, cortical bone sheets 11 can be further cut to uniform size andshape, as in bone-derived sheets 21 of the osteoimplant 20 shown in FIG.2.

FIG. 2 illustrates an osteoimplant 20 comprising cortical bone-derivedsheets 21 having a fully or partially demineralized outer surface withsurface-exposed collagen, and a nondemineralized or partiallydemineralized core 22. Alternatively, one or more bone-derived sheetscan be made from substantially completely demineralized bone. Also,another component such as demineralized bone powder can be coated on thebone-derived sheets. The entire structure has crosslinked collagen onadjacent bone-derived sheets to provide increased adhesion between them.The total thickness of the osteoimplant will ordinarily be at leastabout 2 to about 20 mm. Osteoimplant 20 can be cut, machined, and/orotherwise formed into any other desired shape or dimension forimplantation into a body. Thus, as shown in FIG. 3A, a substantiallycylindrically shaped osteoimplant 30 can be made for use as a long bonesegment replacement 31 for a femur 32 of FIG. 3. To form a cylinder, asubstantially square or rectangular osteoimplant can be shaped on alathe to the required diameter. A cavity can be formed by removing bonematerial with, for example, a drill, or, alternatively, a cavity can beformed by assembling appropriately configured layers of bone-derivedelements.

As shown in FIG. 4, the disc-shaped osteoimplant 40 is shown inserted atthe intervertebral fibrocartilage site 41 on the anterior side ofvertebral column 42.

In FIG. 5, parietal osteoimplant 50 is sized and shaped to form part ofthe parietal bone for skull 51 in FIG. 5A.

In FIG. 6, osteoimplant 60 is built up from bone-derived sheet sections61 of surface demineralized cortical bone, and from bone-derived cubesections 62 of surface demineralized cancellous bone of uniform, squarecross section. These sheet and cube constituents are arranged inalternating layers as shown. After assembly, the structure is subjectedto treatment for crosslinking. Because of the open structure ofosteoimplant 60 resulting from the pattern of channels 63, theosteoimplant permits vascular penetration or host bone ingrowth thereinand/or diffusion of one or more medically/surgical useful substancestherefrom. Osteoimplant 60 is shown installed as a spinal onlay graftattached via insertion of the transverse processes 71 into channels 63,for posterolateral intertransverse process fusion on vertebral column 70of FIG. 7.

In FIG. 8A, osteoimplant 80 comprises bone-derived sheets 81 having afully or partially demineralized outer surface. As shown in FIG. 8, abone-derived sheet has one side coated with tissue transglutaminase 83and, the mating surface of the adjacent sheet is coated with CaCl₂ 82solution. As osteoimplant 80 is assembled, contact between the twocomplimentary sides of bone-derived sheets results in tissuetransglutaminase 83 catalyzing collagen crosslinking at the interface ofadjacent bone-derived sheets 81.

The following examples are further illustrations of the osteoimplant ofthis invention.

EXAMPLE 1

A cortical section of bone from the diaphyseal region was cut in thelongitudinal direction while continuously wetted with water intoapproximately 1.5 mm thick sheets using a diamond-bladed saw. Thecortical bone-derived sheets were then frozen to -70C and freeze-driedfor 48 hours, and subsequently, were placed into excess 0.6N HClsolution for 1.5 hours with constant stirring, washed in water for 5minutes, and soaked for 1.5 hours in BupH phosphate buffered saline. Thebone-derived sheets were assembled into a layered structure and heldwith a clamp. The clamped structure was then placed into a solution of10% neutral, buffered formalin for 48 hours to crosslink the exposedcollagen surfaces. After crosslinking, the clamp was removed, and thestructure was placed in a container and allowed to rinse under runningwater for several hours. The osteoimplant was cut to shape on a bandsaw, and then placed in an excess aqueous solution of glycerol. Afterseven hours, the excess solution was removed, and the osteoimplant wasfreeze-dried.

EXAMPLE 2

Elongate bone-derived fibers were milled from cortical bone, and werefully demineralized in excess 0.6N HCl solution. These fibers werewashed with water, and soaked in an aqueous solution of glycerol.Additionally, fully mineralized bone-derived fibers were added to thesolution which was stirred and left for 12 hours at room temperature.The solution containing the soaked mineralized and demineralizedbone-derived fibers were poured through a 106 micron sieve to recoverthe fibers. The mixture of mineralized and demineralized fibers wasplaced in a cylindrical die, and pressure-treated to 10,000-50,000 psiin a press for 15 minutes, and were then heated for 2 to 12 hours at37-55 degrees C. The resulting osteoimplant pellet was freeze-dried, andplaced in polyethylene glycol diglycidyl ether for 12 hours at roomtemperature.

EXAMPLE 3

Bone-derived sheets derived from human cortical bone, approximately 1 mmthick by 7 mm wide by 50 mm long, were treated for 10 minutes in 0.6NHCl to expose surface collagen. Bone-derived cubes derived from humancancellous bone, 10 mm×10 mm, were treated to expose surface collagen atthe outer borders of the cubes. All bone-derived sheets and cubes werewashed in water. The pieces were assembled together with bone-derivedsheets bordering the cubes, and clamped into place. The construct wasthen placed into a solution of 10% neutral buffered formalin for 3 hoursto crosslink the surface-exposed collagen. The resulting osteoimplantwas then washed in water, and cut to size on a band saw. See FIG. 6.

EXAMPLE 4

Human cortical bone-derived sheets approximately 1 mm thick were surfacedemineralized for 15 minutes in 0.6N HCl, then washed in running water.Tissue transglutaminase was reconstituted to give a 1 mg/ml solution.For each demineralized bone-derived sheet in the construct, the surfacewas blotted dry, then 40 μl/cm² area of the tissue transglutaminase wasapplied to one side and an equivalent volume of 0.1M CaCl₂ solution wasapplied to the mating surface of the next demineralized bone-derivedsheet. This was repeated sequentially. The resulting osteoimplant wasclamped and placed into a humidity chamber to promote crosslinking forapproximately 30 minutes, then washed in water.

EXAMPLE 5

Cortical bone-derived sheets, approximately 2 mm thick, were surfacedemineralized in 0.6N HCl solution for 1 hour with constant stirring.The bone-derived sheets were then coated with dry, demineralized bonepowder having a particle size of 300 microns or less, and assembled intolayers. The construct was clamped into place, and placed into a solutionof 10% neutral buffered formalin for 12 hours to permit collagencrosslinking. The resulting osteoimplant was washed in water to removeexcess chemicals.

What is claimed is:
 1. An osteoimplant which comprises a solid aggregateof bone-derived elements, adjacent bone-derived elements being bonded toeach other through chemical linkages between their surface-exposedcollagen, wherein the chemical linkages are formed by exposing thebone-derived elements to a chemical crosslinking agent, wherein thebone-derived elements are sheets obtained by longitudinally slicing thediaphyseal region of whole cortical bone, and wherein at least one ofthe sheets possesses a fully or partially demineralized outer surfaceand a nondemineralized or partially demineralized core.
 2. Anosteoimplant which comprises a solid aggregate of bone-derived elements,adjacent bone-derived elements being bonded to each other throughchemical linkages between their surface-exposed collagen, wherein thechemical linkages are formed by application of energy, wherein thebone-derived elements are sheets obtained by longitudinally slicing thediaphyseal region of whole cortical bone, and wherein at least one ofthe sheets possesses a fully or partially demineralized outer surfaceand a nondemineralized or partially demineralized core.
 3. Aosteoimplant which comprises a solid aggregate of bone-derived elements,adjacent bone-derived elements being bonded to each other throughchemical linkages between their surface-exposed collagen, wherein thechemical linkages are formed by dehydrothermal treatment, wherein thebone-derived elements are sheets obtained by longitudinally slicing thediaphyseal region of whole cortical bone, and wherein at least one ofthe sheets possesses a fully or partially demineralized outer surfaceand a nondemineralized or partially demineralized core.
 4. Aosteoimplant which comprises a solid aggregate of bone-derived elements,adjacent bone-derived elements being bonded to each other thoughchemical linkages between their surface-exposed collagen wherein thechemical linkages are formed by enzymatic treatment, wherein thebone-derived elements are sheets obtained by longitudinally slicing thediaphyseal region of whole cortical bone, and wherein at least one ofthe sheets possesses a fully or partially demineralized outer surfaceand a nondemineralized or partially demineralized core.
 5. Theosteoimplant of claim 1 wherein the chemical crosslinking agent isselected from the group consisting of monoaldehydes, dialdehydes,polyepoxy compounds, polyvalent metallic oxides, organic tannins,phenolic oxides derived from plants, hydrazide, dicyclohexylcarbodiimide, hexamethylene diisocyanate, sugars and enzymes.
 6. Theosteoimplant of claim 1 wherein the bone-derived elements are exposed tothe chemical crosslinking agent by placing the bone-derived elements ina solution of chemical crosslinking agent.
 7. The osteoimplant of claim1 wherein the bone-derived elements are exposed to the chemicalcrosslinking agent by exposing the bone-derived elements to vapors ofthe chemical crosslinking agent.
 8. The osteoimplant of claim 1 whereinthe chemical crosslinking agent is a polyepoxy compound.
 9. Theosteoimplant of claim 1 wherein the chemical crosslinking agent is amonoaldehyde or dialdehyde.
 10. The osteoimplant of claim 1 wherein thechemical crosslinking agent is formalin.
 11. The osteoimplant of claim 1wherein the chemical crosslinking agent is polyethylene glycoldiglycidyl ether.
 12. The osteoimplant of claim 1 wherein thebone-derived elements are superficially demineralized.
 13. Theosteoimplant of claim 1 further comprising at least one other component.14. The osteoimplant of claim 13 wherein the component is selected fromthe group consisting of reinforcing particles, reinforcing fibers,fillers, bone-growth inducing substances, growth factors, fullymineralized bone, adhesives, plasticizers, flexibilizing agents,cellular material, genetic material, calcification-controlling agents,hydration facilitating agents, biostatic agents, biocidal agents,polymers, inorganic compounds, substances imparting radiopacity andmetallic meshes.
 15. The osteoimplant of claim 1 wherein the solidaggregate of bone-derived elements possesses a compression strength offrom about 10 to about 200 MPa.
 16. The osteoimplant of claim 1 whereinthe solid aggregate of bone-derived elements possesses a compressionstrength of from about 20 to about 200 MPa.
 17. The osteoimplant ofclaim 1 wherein each sheet is approximately 1.5 mm thick.
 18. Theosteoimplant of claim 1 wherein the sheets are assembled into a layeredstructure prior to exposing the sheets to a chemical crosslinking agent.19. The osteoimplant of claim 1 wherein at least one of the sheets isfully demineralized.
 20. The osteoimplant of claim 1 wherein at leastone of the sheets is coated with demineralized bone powder.
 21. Theosteoimplant of claim 18 possessing a total thickness of from about 2 toabout 20 mm.
 22. The osteoimplant of claim 1 configured and dimensionedas a square or rectangle.
 23. The osteoimplant of claim 1 configured anddimensioned as a cylinder.
 24. The osteoimplant of claim 1 wherein thesolid aggregate of bone-derived elements possesses a network of pores,perforations, apertures, channels, or spaces.
 25. The osteoimplant ofclaim 24 wherein the pores, perforations, apertures, channels or spaceshave incorporated therein one or more bone growth inducing or bonehealing substances.
 26. The osteoimplant of claim 2 wherein the energyis heat.
 27. The osteoimplant of claim 2 wherein the energy is radiantenergy.
 28. The osteoimplant of claim 2 wherein the energy isultraviolet light or microwave energy.
 29. The osteoimplant of claim 2wherein the application of energy comprises dye-mediatedphoto-oxidation.
 30. The osteoimplant of claim 2 wherein thebone-derived elements are superficially demineralized.
 31. Theosteoimplant of claim 2 further comprising at least one other component.32. The osteoimplant of claim 31 wherein the component is selected fromthe group consisting of reinforcing particles, reinforcing fibers,fillers, bone-growth inducing substances, growth factors, fullymineralized bone, adhesives, plasticizers, flexibilizing; agents,cellular material, genetic material, calcification-controlling agents,hydration facilitating agents, biostatic agents, biocidal agents,polymers, inorganic compounds, substances imparting radiopacity andmetallic meshes.
 33. The osteoimplant of claim 2 wherein the solidaggregate of bone-derived elements possesses a compression strength offrom about 10 to about 200 MPa.
 34. The osteoimplant of claim 2 whereinthe solid aggregate of bone-derived elements possesses a compressionstrength of from about 20 to about 200 MPa.
 35. The osteoimplant ofclaim 2 wherein each sheet is approximately 1.5 mm thick.
 36. Theosteoimplant of claim 2 wherein the sheets are assembled into a layeredstructure prior to applying energy to the sheets.
 37. The osteoimplantof claim 2 wherein at least one of the sheets is fully demineralized.38. The osteoimplant of claim 2 wherein at least one of the sheets iscoated with demineralized bone powder.
 39. The osteoimplant of claim 36possessing a total thickness of from about 2 to about 20 mm.
 40. Theosteoimplant of claim 2 configured and dimensioned as a square orrectangle.
 41. The osteoimplant of claim 2 configured and dimensioned asa cylinder.
 42. The osteoimplant of claim 2 wherein the solid aggregateof bone-derived elements possesses a network of pores, perforations,apertures, channels, or spaces.
 43. The osteoimplant of claim 42 whereinthe pores, perforations, apertures, channels or spaces have incorporatedtherein one or more bone growth inducing or bone healing substances. 44.The osteoimplant of claim 3 wherein the bone-derived elements aresuperficially demineralized.
 45. The osteoimplant of claim 3 furthercomprising at least one other component.
 46. The osteoimplant of claim 3wherein the component is selected from the group consisting ofreinforcing particles, reinforcing fibers, fillers, bone-growth inducingsubstances, growth factors, fully mineralized bone, adhesives,plasticizers, flexibilizing agents, cellular material, genetic material,calcification-controlling agents, hydration facilitating agents,biostatic agents, biocidal agents, polymers, inorganic compounds,substances imparting radiopacity and metallic meshes.
 47. Theosteoimplant of claim 3 wherein the solid aggregate of bone-derivedelements possesses a compression strength of from about 10 to about 200MPa.
 48. The osteoimplant of claim 3 wherein the solid aggregate ofbone-derived elements possesses a compression strength of from about 20to about 200 MPa.
 49. The osteoimplant of claim 3 wherein each sheet isapproximately 1.5 mm thick.
 50. The osteoimplant of claim 3 wherein thesheets are assembled into a layered structure prior to subjecting thesheets to dehydrothermal treatment.
 51. The osteoimplant of claim 3wherein at least one of the sheets is fully demineralized.
 52. Theosteoimplant of claim 3 wherein at least one of the sheets is coatedwith demineralized bone powder.
 53. The osteoimplant of claim 50possessing a total thickness of from about 2 to about 20 mm.
 54. Theosteoimplant of claim 3 configured and dimensioned as a square orrectangle.
 55. The osteoimplant of claim 3 configured and dimensioned asa cylinder.
 56. The osteoimplant of claim 3 wherein the solid aggregateof bone-derived elements possesses a network of pores, perforations,apertures, channels, or spaces.
 57. The osteoimplant of claim 56 whereinthe pores, perforations, apertures, channels or spaces have incorporatedtherein one or more bone growth inducing or bone healing substances. 58.The osteoimplant of claim 4 wherein the enzymatic treatment comprisestissue transglutaminase.
 59. The osteoimplant of claim 4 wherein thebone-derived elements are superficially demineralized.
 60. Theosteoimplant of claim 4 further comprising at least one other component.61. The osteoimplant of claim 60 wherein the component is selected fromthe group consisting of reinforcing particles, reinforcing fibers,fillers, bone-growth inducing substances, growth factors, fillymineralized bone, adhesives, plasticizers, flexibilizing agents,cellular material, genetic material, calcification-controlling agents,hydration facilitating agents, biostatic agents, biocidal agents,polymers, inorganic compounds, substances imparting radiopacity andmetallic meshes.
 62. The osteoimplant of claim 4 wherein the solidaggregate of bone-derived elements possesses a compression strength offrom about 10 to about 200 MPa.
 63. The osteoimplant of claim 4 whereinthe solid aggregate of bone-derived elements possesses a compressionstrength of from about 20 to about 200 MPa.
 64. The osteoimplant ofclaim 4 wherein each sheet is approximately 1.5 mm thick.
 65. Theosteoimplant of claim 4 wherein the sheets are assembled into a layeredstructure prior to subjecting the sheets to enzymatic treatment.
 66. Theosteoimplant of claim 4 wherein at least one of the sheets is fullydemineralized.
 67. The osteoimplant of claim 4 wherein at least one ofthe sheets is coated with demineralized bone powder.
 68. Theosteoimplant of claim 65 possessing a total thickness of from about 2 toabout 20 mm.
 69. The osteoimplant of claim 4 configured and dimensionedas a square or rectangle.
 70. The osteoimplant of claim 4 configured anddimensioned as a cylinder.
 71. The osteoimplant of claim 4 wherein thesolid aggregate of bone-derived elements possesses a network of pores,perforations, apertures, channels, or spaces.
 72. The osteoimplant ofclaim 71 wherein the pores, perforations, apertures, channels or spaceshave incorporated therein one or more bone growth inducing or bonehealing substances.